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A kind of preparation method of anticoagulant peptide and its prepared small peptide and application

An anticoagulant peptide and anticoagulant technology, which is applied in the field of health care products and medicine, can solve the problems of high drug price, low curative effect, and high economic pressure, and achieve simple preparation methods, good anticoagulant activity, and high The effect of added value

Inactive Publication Date: 2021-02-12
GUANGDONG OCEAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The former has low curative effect and large side effects; although the latter has high curative effect and few side effects, the price of the drug is high and it can only be administered by injection, which is cumbersome and economically stressful

Method used

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  • A kind of preparation method of anticoagulant peptide and its prepared small peptide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Take a certain amount of buffalo milk in a 50mL test tube, put it in a high-speed centrifuge and centrifuge at 6000r / min, take it out after 15 minutes, remove the upper fat layer, and obtain skimmed buffalo milk. After skimming the milk, adjust the pH to about 10 and boil for 10 minutes, let it cool to room temperature, take 10.0 mL of the treated skim buffalo milk in an Erlenmeyer flask, add the enzyme solutions in the examples, and add 5 mL of distilled water, and use 0.1 mol / L The pH adjusted by NaOH or HCl is the pH, temperature and enzymolysis time in the examples respectively; after the water (enzyme) hydrolysis is completed, the enzyme is inactivated and the supernatant is obtained by centrifuging at 5000r / min for 20min to measure the antithrombin activity.

[0026] Wherein the composite enzymatic hydrolysis condition in embodiment 1~9 is shown in table 1:

[0027] Among them, the method of mixed enzymolysis was adopted in Examples 7~9. Taking Example 7 as an ...

Embodiment 10

[0030] Examples 10-12 were carried out by compound enzymatic hydrolysis, as shown in Table 2 for details. Taking Example 10 as an example, first, papain was used in an amount of 6000u / mL at 60°C, pH 5.0, and enzymatic hydrolysis for 2 hours Finally, inactivate the enzyme; then add alkaline protease according to the dosage of 6000u / mL at 50℃, the pH is 8.0, after 4 hours of enzymolysis, inactivate the enzyme; finally add neutral protease according to the dosage of 6000u / mL at 40℃, the pH is 6.0, enzymatic hydrolysis for 2 hours, so as to complete the entire enzymatic hydrolysis process.

[0031] The experimental condition of table 2 embodiment 10~12

[0032]

Embodiment 13

[0033] Example 13: Determination of antithrombin activity of hydrolyzate by improved thrombin titration method

[0034] 1. Configure different concentrations of thrombin solutions

[0035] Prepare 0.05 mol / L Tris-HCl solution containing 0.05 mol / L NaCl, pH7.4, to prepare 0.5% fibrinogen. Prepare thrombin solutions with different concentration gradients: 20NIH / mL (as a reference concentration, that is, 1 time), and 5 μL of this solution is a titration volume, that is, 1V, which is 0.1 ATU:

[0036] 20 NIH÷(1000μL÷5μL)=20NIH÷200=0.1NIH, because 1NIH=1ATU, so 0.1 ATU=0.1NIH, 20 NIH / mL (as a reference concentration, 1 time), 40 NIH / mL (2 times ), 100 NIH / mL (ie 5 times), etc.

[0037] 2. Determination of anticoagulant activity of hydrolyzate

[0038] Take several small test tubes of 7.5 mm × 100 mm, add 200 μL of 0.5% fibrinogen to each, and then add 50 μL of hydrolyzed extraction solution to each, and place them in a constant temperature water bath at 37°C for 5 minutes.

[0...

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Abstract

The invention discloses a method for preparing an anticoagulant peptide. The milk is centrifuged, the fat layer is removed, and the pH is adjusted to 5.0-8.0 to carry out the enzymolysis reaction; the temperature of the enzymolysis reaction is 40-60°C, and the enzymolysis time is 2~4h; the ratio of material to water for enzymolysis is 1:(1.5~3); the type of enzyme required for the enzymolysis reaction is one or more of papain, neutral protease and alkaline protease. The preparation method provided by the invention has simple process and easy-to-obtain raw materials. The prepared small peptide substances have good anticoagulant activity and can be used to make up for the defects of heparin drugs. The invention uses buffalo milk as the preparation source, and the side effects At the same time, it has opened up a new way of processing fresh milk resources and improved the high added value of milk, which is of great significance to the research in the fields of medicine and food.

Description

technical field [0001] The invention belongs to the technical field of medicine and health products, and more specifically relates to a preparation method of an anticoagulant peptide, and the prepared small peptide and its application. Background technique [0002] Thrombus refers to the formation of a semi-clot-like substance on the surface of blood vessels or the intima of the heart during the flow of blood components, which is composed of insoluble fibrin, deposited platelets, accumulated white blood cells, and trapped red blood cells. Thromboembolic disease is a common cardiovascular and cerebrovascular disease. The stenosis and occlusion of the blood vessel lumen caused by thrombus will cause ischemia and infarction of major organs and cause dysfunction. It often manifests as myocardial infarction, ischemic cerebral infarction and Venous thromboembolism. [0003] Thromboembolic disease is known as the invisible killer of cardiovascular and cerebrovascular diseases. It ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/06C07K14/47A61K38/01A61P7/02A61P9/00A61P9/10
CPCA61K38/00C07K14/4732C12P21/06
Inventor 苗艳丽李泳谷长生黄娜张兆霞邓春梅廖铭能
Owner GUANGDONG OCEAN UNIVERSITY
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